Team:Paris/August 28

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='''Transformation of OmpR*'''=
='''Transformation of OmpR*'''=
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='''Construction of pFlhB - mRFP Tripart (LVA+)'''=
 
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Aim : Construction of ''' "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078)''' [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 
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We can only do the construction with mRFP Tripart (LVA+) because the stable strain with the Biobricks I732011 (mRFP Tripart LVA-) don't to growth.
 
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==Digestion==
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=Cloning of EnvZ*=
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==='''Measurement of the concentration of D187 purified'''===
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[[Team:Paris/Notebook/Protocols#Concentration_of_the_Miniprep | Protocol (it's same that for Miniprep)]]
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The cloning of EnvZ* didn't worked. So we decided to try one more time, starting from the PCR product.
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{|border="1" style="text-align: center"
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==Digestion==
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|'''Digestion Name'''
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|'''Concentration (µg/mL)'''
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|'''Ratio 260/280'''
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|D187
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|}
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==Ligation==
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[[Image:EnvZ pSB1A2.JPG|thumb|Digestion of EnvZ* and lasR-pSB1A2]]
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[[Team:Paris/Notebook/Protocols#Ligation |Protocol]]
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{|border="1" style="text-align: center"
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|'''Ligation Name'''
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|'''Vector Name'''
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|'''Volume Vector (µL)'''
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|'''Insert'''
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|'''Volume Insert (µL)'''
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|-
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|L168
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|D187
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|D186
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|-
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|Control L168
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|D166
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| -
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|}
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=Cloning of EnvZ*=
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==Digestion==
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{|border="1" style="text-align: center"
{|border="1" style="text-align: center"
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|XbaI & PstI
|XbaI & PstI
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|}
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<br>
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'''Purification'''
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*EnvZ*: directly by the Qiagen kit
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*pSB1A2: excision of the band of interest (2 kb) and then purification using the Qiagen kit
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=Freezer empty=
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[[Team:Paris/Notebook/Protocols#Digestion |Protocol]]
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We did again [[Team:Paris/Notebook/Freezer#Digestion Products |D110]] and  [[Team:Paris/Notebook/Freezer#Digestion Products |D125]].

Latest revision as of 19:09, 9 September 2008

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Contents

Transformation of OmpR*

Cloning of EnvZ*

The cloning of EnvZ* didn't worked. So we decided to try one more time, starting from the PCR product.

Digestion

Digestion of EnvZ* and lasR-pSB1A2
Digestion n° Name volume of DNA digested total volume reaction enzymes used
D159 PCR147 (EnvZ*) 3 µL 30 µL XbaI & PstI
D116 MP108 (lasR-pSB1A2) 2 µL 30 µL XbaI & PstI


Purification

  • EnvZ*: directly by the Qiagen kit
  • pSB1A2: excision of the band of interest (2 kb) and then purification using the Qiagen kit

Freezer empty

Protocol

We did again D110 and D125.

Retrieved from "http://2008.igem.org/Team:Paris/August_28"